RNF213通过泛素化mTOR调控自噬在脑缺血再灌注损伤中的作用及机制

RNF213基因被发现参与多种脑血管病的病理生理，然而，RNF213是否参与脑缺血再灌注损伤仍存在知识空白。我们的前期研究发现缺血再灌注损伤神经元会产生大量RNF213蛋白，敲除RNF213基因可抑制自噬、上调mTOR、减小脑梗死体积，但具体机制不明。本项目拟在动物水平及细胞水平，利用RT-qPCR、免疫荧光、Western Blot、电镜多维度评估缺血再灌注神经元RNF213与自噬水平，佐以病毒技术构建RNF213过表达与基因沉默小鼠，通过已构建的RNF213基因敲除小鼠，比对自噬水平、脑梗死体积及肢体功能预后，进一步检测mTOR是否存在泛素化修饰，明确RNF213是否通过泛素化mTOR激活自噬继而加重缺血再灌注神经元损伤，以期为脑缺血再灌注损伤提供新的干预靶点。

RNF213 gene was found to be involved in the pathophysiology of a variety of cerebrovascular diseases. However, there are still critical knowledge gaps regarding the roles of RNF213 in cerebral ischemia-reperfusion injury. Our preliminary basis confirmed that neurons with ischemia-reperfusion injury will produce a large amount of RNF213 protein, and RNF213 knockout can inhibit autophagy, upregulate mTOR, reduce cerebral infarction volume, but its specific mechanism is unclear. This project is to evaluate the expression of RNF213 and autophagy by RT-qPCR, immunofluorescence, Western Blot and electron microscope in vitro and in vivo. In addition, autophagy-related proteins, cerebral infarction volume and limb function are compared among RNF213-overexpressed mouse, RNF213-silencing mouse and RNF213-knockout mouse. What’s more, we would further detect whether there is ubiquitination modification in mTOR. This study is to clarify whether RNF213 activates autophagy through ubiquitination of mTOR and then aggravates ischemia-reperfusion neuronal damage, and then provide novel intervention targets for ischemic-reperfusion injury.